The present invention was made with support of National Cancer Institute grant number CA 33895. The United States Government may have rights in the invention.
The invention relates generally to cell surface carbohydrate markers, and more specifically to a method of detecting T-cell dysfunctions using a specific hexasaccharide structure found on the protein leukosialin.
The hematopoietic system can be divided into two major cell lineages. The first consists of the myeloid cell lineage and is responsible for functions such as oxygen exchange, destruction of invading bacteria, modulation of allergic inflammatory reactions and blood clotting. Cells of the myeloid lineage which mediate these functions include erythrocytes, granulocytes, monocytes, eosinophils and platelets.
The second major lineage within the hematopoietic system consists of the lymphoid cell lineage. This class is primarily responsible for host immunity and includes B-lymphocytes (B-cells) and T-lymphocytes (T-cells). B-cells are responsible for antibody-mediated immunity. Antibodies produced by B-cells circulate in the blood stream and are capable of binding and neutralizing a foreign antigen. T-cells, on the other hand, function in cell-mediated immunity. They are involved in a wide variety of immune functions, all of which involve the direct interaction of the T-cell with some other cell. The interacting cell can be another T-lymphocyte, a B-lymphocyte, a macrophage or a target cell. In the latter case, the antigen to be recognized is generally found at the surface of a target cell. Types of T-lymphocytes include, for example, helper T-cells, suppressor T-cells and cytotoxic T-cells.
The ability of the immune system to perform both antibody-mediated and cell-mediated immunity is the basis of our defense against invading pathogenic organisms. These two types of immune response can be dissociated to a large extent but not without compromising the host's ability to provide complete protection. For instance, in humans, congenital agammaglobulinemia is a disease in which antibody-mediated immunity is deficient while cell-mediated immunity is normal. In contrast, congenital thymus deficiencies produce individuals with greatly impaired cell-mediated immunity but relatively high serum antibody levels.
In many instances, an individual cannot survive without the function of both immunities. For example, loss of T-cell function due to cancers such as acute T-lymphocytic leukemia (T-ALL) can eventually lead to death. In this disease, the normal population of T-cells is overtaken by the faster growing malignant population until complete loss of normal T-cell-mediated immunity occurs. Acquired Immune Deficiency Syndrome (AIDS) is another disease where complete loss of T-cell function results in death. AIDS is caused by infection with a retrovirus. An analogous example of a congenital T-cell immunodeficiency is Wiscott-Aldrich Syndrome (WAS).
Cell surface carbohydrate structures have been used as tumor or differentiation markers since they are known to undergo characteristic changes during differentiation. For example, leukemic cells have been classified by their surface glycoprotein molecular weight patterns with respect to their relationship to normal blood cells (Gahmberg and Andersson, Annals New York Academy of Sciences 240-255 (1978). For a detailed review of such onco-differentiation markers as they apply to the myeloid cell lineage, see Fukuda, Biochimica et Biophysica Acta 780:119-150 (1985), which is incorporated herein by reference.
One of the most extensively studied markers is a major sialoglycoprotein known as leukosialin, also known as sialophorin or CD43. It is an integral membrane protein which carries one N-linked and eighty O-linked carbohydrate chains. Its mobility on SDS-PAGE depends largely on the structures of the O-linked glycans and the extent of sialiation. This glycoprotein has been shown to be differentially glycosylated between different leukemic cell lines and to increase in molecular weight upon activation of normal human T-cells, Carlsson et al., The Journal of Biological Chemistry 261:12787-12791 (1986), and Piller et al., The Journal of Biological Chemistry 263:15146-15150 (1988). The differential glycosylation between leukemic cell lines is due to characteristic sets of O-linked oligosaccharides whereas the increase in molecular weight upon activation is due exclusively to the expression of a branched hexasaccharide structure. Resting T-cells express on leukosialin the tetrasaccharide NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3(Neu NAc.alpha.2.fwdarw.6)GalNAc-Ser/Thr but activated T-cells carry the more complex structure NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3(Neu NAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.6)GalNAc-Ser/T hr. This branched hexasaccharide structure has also been found on leukosialin from T-lymphocytes isolated from WAS patients, Piller et al., J. Cell Biol. 108:191a (1989), and on cell surface glycoproteins of the myeloid cell lineage, Fukuda et al., The Journal of Biological Chemistry 261:12796-12806 (1986), both of which are incorporated herein by reference.
The shift in biosynthesis of O-glycans in activated T-cells compared to resting T-cells is apparently caused by a stimulation of the .beta.1.fwdarw.6GlcNAc-transferase (Core 2 GlcNAc-T). Activated T-cells also exhibit a concomitant decrease in .alpha.2.fwdarw.6 sialyltransferase activity. This shift in glycosyltransferase activity results in a decrease in the amount of tetrasaccharide expressed on leukosialin paralleled by an increase in the amount of expressed hexasaccharide, Piller et al., (1988) supra.
Despite the increased interest in the elucidation of cell surface structures and their biosynthetic mechanisms, very few markers have been discovered which accurately predict pathological disorders of cell-mediated immunity. Leukosialin has been extensively characterized as a marker for normal T-cell activation. However, there has been no indication that this determinant can also be used as a marker for pathologic T-cell dysfunctions.
There thus exists a need for a method which can accurately diagnose T-cell dysfunctions. The present invention satisfies this need and provides related advantages as well.